Method for applying, on a solid support, at least one binding partner to a molecule

ABSTRACT

A process for applying, in a tubular solid support, at least one binding partner P1 to an analyte to be detected or quantified in a test sample, including steps: (i) connect the support to a suction-discharge device, (ii) draw up into the support, by one of its ends, a solution including the binding partner P1, called sensitization solution S1, contained in a container, called container C1, (iii) continue contact between solution S1 and the inner surface of the support for a time between 0 s and 11 min, (iv) discharge the solution S1 into a container, which is optionally the container C1, steps (ii) to (iv) forming a cycle that can be repeated at least once, over a total duration of at least 1 min and at most 2.5 h. Also, the use of the coated support for the detection or quantification of an analyte in a test sample.

The present invention relates to the field of in vitro detection ofanalytes in test samples likely to contain these analytes. Inparticular, the invention relates to a novel process for applyingbinding partners to the desired analyte on a tubular solid support,which support is then used for the detection of the analyte.

In the field of analysis of test samples likely to contain analytes ofinterest, it is known to use methods based on specific measurements suchas signal measurements. The analysis of the test sample may include theuse of a binding partner to the analyte to be detected or quantified inthe sample. The binding partner makes it possible to isolate the analyteto be tested in order to implement its detection by obtaining a reactionproduct emitting a signal such as a colored signal or a fluorescencesignal.

The binding partner used to isolate the analyte from the test sample ismost often bound to the surface of a solid support. Different solidsupports can be used such as solid supports, such as beads, hollowsupports having a bottom, called closed hollow supports or hollowsupports closed at one end, such as wells, or bottomless hollowsupports, called open hollow supports or hollow supports open at eachend, in the form of tubes, such as cones or pipettes.

When the supports are hollow, open or closed supports, the bindingpartners to the analyte used are fixed, for example by adsorption (it isthen said that they are coated), inside the support, on its innersurface.

Usually, the application of binding partners to the inner surface ofhollow supports is made by placing a solution containing the bindingpartners on the inner surface of the support in static form for a longperiod of more than 6 hours, and on average overnight, as described inpatent application FR2417094. Because of this long preparation time,biological analysis using these media cannot be carried out quicklybecause it is necessary to wait at least the time for the preparation ofthe media and, in most cases, the preparation of the media and theanalysis must be carried out over at least two days. Indeed, for abiological analysis to be acceptable, particularly in in vitrodiagnostics, and especially in cases where an analysis report is urgent,it is necessary that the total analysis time be as short as possible.

As a result, as a matter of routine, the substrates are prepared inadvance, dried and then stored in desiccant bags to avoid any stabilityproblems. They are then used with other elements necessary for theanalysis, such as reagents.

The application of the binding partners on the surface of the hollowsupports is therefore done over a long period of time, generally using asingle solution containing these binding partners, called thesensitization or coating solution, in a closed system, i.e. the samesolution whose concentration of binding partners is always used whenthey are applied on the inner surface of the hollow supports. It is thennecessary for the solution to contain these binding partners in largeexcess, which is costly. Nevertheless, the biggest disadvantage withthis method is the contact time required to obtain the right amount ofapplied binding partner.

Another process for applying binding partners in closed hollow supports,such as microplates, has been developed. It consists of bringingtogether a sensitization solution containing the binding partners, thenpossibly shaking the plate by means of a vibration or orbital motionshaking plate for several hours, on average 4 hours. Even if the coatingtime is a little shorter than a static process, it is far fromacceptable. In addition, again, since shaking takes place in a closedsystem, as with static application, it is necessary that thesensitization solution contains the binding partners in large excess.Another disadvantage of this process is that it cannot be used with openhollow supports. Finally, the disadvantage of this process is that thesupports thus coated by the binding partners are not very reproduciblebecause the process cannot be automated.

Compared to hollow supports closed at one end, the advantage of hollowsupports open at each end is that they can also be used for pipettingand transporting reaction liquids, including the sample. They are usedin particular to draw up liquids and then discharge them.

An example of a system using such an open hollow support in which thebinding partners are applied is the VIDAS® system (bioMérieux). Thisinstrument uses a pipette tip+test strip pair for the analysis of testsamples. The test strip, introduced into the system, consists of severalcuvettes, some of which are filled with predetermined liquids, usefulfor analysis. The test strip also includes a cuvette adapted to receivea sample to be tested and a cuvette for reading the test result. Thus,in this system, the pipette tip contains part of the reagents necessaryfor the emission of the signal and the test strip contains the otherpart of the reagents. When analyzing this test sample, the pipette tipis used to draw a quantity of said sample and deposit said sample insidethe different cuvettes present in the test strip. The liquids presentinside the different cells can react with the sample to obtain, at theend of a liquid transfer cycle, for example from one cell to another, aliquid, or reaction medium, on which the measurements of a fluorescencesignal are made. If the analyte to be detected or quantified is presentin the test sample, it will bind to the binding partner present on thesurface of the pipette tip, and then the liquids present in thedifferent cuvettes will be used to detect this analyte. With the VIDAS®instrument, the binding partners are applied in the pipette tipsbeforehand for about 12 hours, statically, so that the kits sold containpre-coated pipette tips and are stored for later use.

Regardless of how the binding partner is applied and regardless of themedium, this system requires the use of at least two instruments ofdifferent nature and functionality, one to apply the capture partnersand the other to perform the analysis.

The present invention therefore aims to overcome the disadvantages ofthe state of the art by proposing a process that dynamically and rapidlyapplies one or more binding partners to the inner surface of an openhollow support, thus significantly shortening the application time ofthe binding partners. This way, it is possible to immediately continuethe analysis, thus avoiding the storage of the coated substrate.Moreover, with such a process, a single type of instrument may besufficient for both the application of the binding partner(s) and theanalysis, even if these two steps are not implemented immediately oneafter the other. Finally, having such a process, which significantlyshortens the application time of the binding partner(s) and uses asensitization solution in which the number of binding partners isreduced, also allows for significant economic savings when producingopen hollow supports.

To achieve such an objective, the subject matter of the inventionrelates to a process for applying, in a tubular solid support,optionally flared, having a circular or ellipsoidal opening at each end,such as a pipette tip or a pipette, at least one binding partner P1 toan analyte to be detected or quantified in a test sample, comprising thefollowing steps:

(i) connect the solid support to a suction-discharge device,(ii) draw up into the support, by one of its ends, a solution comprisingsaid at least one binding partner P1, called sensitization solution S1,contained in a container, called “container C1”,(iii) continue contact between the sensitization solution S1 and theinner surface of the solid support for a time between 0 s and 11 min,(iv) discharge the sensitization solution P1 into a container, which isoptionally said container C1,

steps (ii) to (iv) forming a cycle that can be repeated at least once,over a total duration of at least 1 min and at most continue contactbetween 2.5 h.

The process according to the invention has in combination one and/orother of the following features:

the contact time of step (iii) between the sensitization solution S1 andthe inner surface of the solid support is between 2 s and 1 min,preferably between 5 s and 25 s, more preferably between 6 s and 20 s;

steps (ii) to (iv) are repeated 10 to 150 times, preferably 35 to 80times;

the total of the repeated cycles is between 10 and 20 min and ispreferably 15 min;

the following steps (v) to (vii) are implemented when the suction(ii)/contact (iii)/discharge (iv) cycles are completed:

(v) draw up into the support in which the at least one binding partnerP1 is applied, a wash solution W1 contained in a container calledcontainer CW1,(vi) continue contact between the wash solution W1 and the inner surfaceof the solid support for a time between 0 s and 5 min,(vii) discharge the wash solution W1 into a container, which may or maynot be said container CW1,

steps (v) to (vii) forming a cycle that can be repeated at least once,over a total duration of at least 1 min and at most 2.5 h;

the sensitization solution S1 also contains at least one binding partnerP2 to the binding partner P1 to the analyte.

The binding partner P2 to the binding partner P1 to the analyte to bedetected or quantified can advantageously be applied to the innersurface of the solid hollow support according to the same process of theinvention as described above by repeating the following steps:

(a) connect the solid support to a suction-discharge device,(b) draw up into the solid support, by one of its ends, a solutioncomprising said at least one binding partner P2, called sensitizationsolution S2, contained in a container, called container C2,(c) continue contact between the sensitization solution S2 and the innersurface of the solid support for a time between 0 s and 11 min,(d) discharge the sensitization solution S2 into a container, which isoptionally said container C2,steps (b) to (d) forming a cycle that can be repeated at least once,over a total duration of at least 1 min and at most 2.5 h.

The process for applying the partner P2 has in combination one and/ormore of the following features:

it also includes the following steps (e) to (g), implemented when thesuction (b)/contact (c)/discharge (d) cycles are completed:

(e) draw up into the support in which the at least one binding partnerP2 is applied, a wash solution W2 contained in a container calledcontainer CW2,

(f) continue contact between the wash solution W2 and the inner surfaceof the solid substrate for a time of between 0 s and 5 min,

(g) discharge the wash solution W2 into a container, which is optionallysaid container CW2,

steps (e) to (g) forming a cycle that may be at least once, over a totalduration of at least 1 min and at most 2.5 h;

it also includes the application, in said solid support, after thesuction (b)/contact (c)/discharge (d) cycles, of said at least onebinding partner P1;

the application of said at least one binding partner P1, whenimplemented, is carried out according to an application process of theinvention, as described above.

The open hollow solid support, coated with the analyte binding partnerP1, optionally via the binding partner P1 to P2, is advantageously usedfor the detection or quantification of the analyte contained in a testsample. Also, another subject matter of the invention relates to aprocess for the in vitro detection or quantification of an analyte in atest sample likely to contain said analyte, said process using at leastone tubular solid support, optionally flared, having a circular orellipsoidal opening at each end, such as a pipette tip or a pipette, inwhich at least one binding partner P1 to an analyte to be detected orquantified in the test sample is applied according to the applicationprocess of the invention, which process of detection or quantificationcomprises the steps of contacting the test sample with the solid supportand detecting the binding, if the analyte is present, of said analyteand said at least one binding partner P1.

The detection or quantification process has in combination one or moreof the following features:

-   -   the binding partner P1 is an immunoassay partner;    -   the detection of the binding of said analyte is carried out by a        sandwich test using another binding partner to the analyte,        optionally of a different nature, which is labelled, or by a        competitive test using a labelled compound competing with the        analyte to be detected or quantified;    -   the detection of the analyte is carried out using an enzyme and        an enzyme substrate catalyzed by said enzyme, preferably        alkaline phosphatase and 4-methylumbelliferyl phosphate;    -   the steps of contacting the test sample with the solid support        and detecting the binding, if the analyte is present, of said        analyte and said at least one binding partner P1 are carried out        immediately after the process of applying at least one binding        partner P1 in said solid support as defined above, preferably        with the same instrument or an instrument of the same range;    -   the steps of contacting the test sample with the solid support        on which said at least one binding partner P1 is applied, and        detecting the binding of said analyte and said at least one        binding partner P1, include the following steps consisting in:    -   (1) drawing up into said solid support, by one of its ends, said        sample contained in a container, called container CE, leaving in        contact and discharging said sample into a container, which is        optionally said container CE,    -   (2) drawing up into said solid support, at the same end, a        solution comprising a compound conjugated to a label, such as        another binding partner to the analyte or a compound competing        with the analyte, called conjugate solution CS, said conjugate        solution CS being contained in a container called container CC,        allowing said conjugate solution CS to contact and discharge        into a container, which is optionally said container CC,    -   (3) if the label uses a detection substrate, drawing up into        said solid support, at the same end, a solution comprising a        detection substrate with which the label will react, called        substrate solution SS, said substrate solution SS being        contained in a container called container CS, leaving in contact        and discharging said substrate solution into a container, which        is optionally said container CS, and    -   (4) measuring the transmitted signal.

Of course, the process can include one or more washing steps betweensteps (1) and (2) and steps (2) and (3), implemented as described below.

Application in an open hollow solid support of at least one bindingpartner means that the at least one binding partner is fixed inside thesupport by any means known to the skilled person such as adsorption,covalent binding, hydrogen binding, electrostatic binding and ionicbinding, etc. The type of fixing of the binding partners inside thehollow support depends on the nature of the support and in particularits surface. Thus, the support can be made of plastic, such aspolycarbonate, styrene-butadiene copolymers, polymers (polystyrene,polypropene, polypropylene, polyethylene, etc.) or stainless steel, withor without pretreatment of the inner surface. The inner surface of thehollow support may have been functionalized by reactive groups that willresult in the formation of a covalent bond with the binding partner. Theinner surface of the hollow support may also have undergone treatmentsto improve the fixation of the binding partners, such as ionizingradiation treatment such as irradiation with X-rays, acceleratedelectrons or gamma radiation. Such pretreated or untreated supports arecommercially available, such as from Thermofisher.

Analyte to be detected or quantified means any molecule representativeof the presence of microorganisms or a disease to be detected,characterized or monitored. It also includes the screening of drugs orphysical-active substances, drugs and the follow-up of treatments inpatients. Preferably, the detection or quantification of this analyte isperformed by an immunoassay which is a test widely known to the skilledperson, involving immunological reactions between this analyte to bedetected and a binding partner(s) to this analyte. Therefore, examplesof analytes include antibodies, for example autoantibodies or antibodiesagainst pathogen proteins, for example viruses such as HIV, SIV, FIV,HCV, HBV, HAV, HEV, VZV, CMV, EBV, HSV1, HSV2, bacteria such asMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae,Borrelia burgdorferi strict sensu, Borrelia afzelii, Borrelia garinii,Borrelia spielmanii, Clostridium difficile, Clostridium botulinum,salmonella, Klebsiella, Legionella, Proteus, Klebsiella, Escherichiacoli, Shigella, Pseudomonas aeruginosa, Staphylococcus aureus, Treponemapallidum, yeasts such as Candida albicans, fungi such as Aspergillusfumigatus, Mucorales, etc.), proteins different from antibodies, forexample biomarkers such as procalcitonin (PCT), thyroid hormones (forexample TSH), cardiac troponins (TnI, TnT, and associated complexes),BNP, NT-proBNP, D-dimer, CRP, S100B, L-FABP, H-FABP, cancer markers suchas CA-15-3, CA-19-9, etc., or pathogen proteins, peptides, for examplepathogen protein fragments as described above, and haptens, for examplesteroids or vitamins.

The analyte is likely to be contained in a test sample that may be ofvarious origins, such as food, environmental, biological, veterinary,clinical, pharmaceutical or cosmetic origin.

Food samples include, but are not limited to, a sample of milk products(yogurts, cheeses, etc.), meat, fish, eggs, fruit, vegetables, water,beverages (milk, fruit juices, soda, etc.). Of course, these foodsamples may also come from more elaborate sauces or dishes or fromunprocessed or partially processed raw materials. A food sample may alsobe obtained from animal feed, such as oilcake or animal meal. All thesesamples, if they are not liquid, are preprocessed to be in liquid form.

As indicated above, the sample may be of environmental origin and mayconsist, for example, of a surface sample, water, air, etc.

The sample may also consist of a biological sample, of human or animalorigin, which may consist of samples of biological fluid (urine, wholeblood or derivatives such as serum or plasma, saliva, pus, cerebrospinalfluid, etc.), stool (for example cholera diarrhea), nose, throat, skin,wounds, organs, tissues or isolated cells, swab samples. This list isobviously not exhaustive.

In general, the term “sample” refers to a part or quantity, moreparticularly a small part or quantity, taken from one or more entitiesfor analysis. This sample may have undergone prior treatment, involvingfor example mixing, dilution or grinding steps, particularly if thestarting entity is in a solid state.

Binding partner P1 to the analyte to be detected or quantified in a testsample means any partner capable of binding to said analyte. Of course,the concepts of analyte and binding partner are quite distinct from eachother. The analyte is the compound present in the test sample, while thebinding partner P1, which is applied according to the process of theinvention, is the compound that will bind to the analyte. The nature ofthe binding partner P1 depends on the nature of the analyte and the typeof test used. Preferably, the test will be an immunoassay.

The term “immuno” in “immunoassay” for example, is not to be consideredin this application as strictly indicating that the binding partner isan immunological partner, such as an antibody. Indeed, the skilledperson also uses this term widely when the binding partner, also calledligand, is not an immunological partner but is for example a receptorfor the analyte to be measured. Thus, it is known to speak of theenzyme-linked immunosorbent assay (ELISA) for assays that usenon-immunological binding partners, more broadly called ligand bindingassays, which could be restated as assays using ligand binding, whereasthe term “immuno” is included in the acronym ELISA. For the sake ofclarity, the Applicant will use the term “immuno” throughout theapplication for any test or assay using a binding partner, even when itis not an immunological partner.

Examples of immunoassay binding partners P1 include binding partners ofan immunological nature or origin such as antibodies (monoclonal orpolyclonal) and antibody fragments (such as Fab, Fab′, F(ab′)2), scFv(single chain variable fragment), dsFv (double-stranded variablefragment), well known to the skilled person, as well as binding partnersthat are not of an immunological nature or origin such as proteinsdifferent from antibodies, peptides, oligonucleotides, nanofitins,analyte receptors if they exist, aptamers, DARPins or any other moleculethat is known to have an interaction with said analyte.

Nanofitins (trade name) are small proteins that, like antibodies, areable to bind to a biological target, allowing it to be detected,captured or simply targeted within an organism.

Aptamers are oligonucleotides, usually RNA or DNA, identified in bankscontaining up to 1015 different sequences, by an in vitro combinatorialselection method called SELEX for “Systematic Evolution of Ligands byExponential Enrichment” (Ellington A D and Szostak J W., 1990). Mostaptamers are composed of RNAs, due to the ability of the RNA to adoptvaried and complex structures, which allows the creation of cavities ofvarious geometries on its surface, allowing various ligands to beattached. These are biochemical tools of interest that can be used inbiotechnological, diagnostic or therapeutic applications. Theirselectivity and ligand binding properties are comparable to those ofantibodies.

Designed ankyrin repeat proteins (DARPins) (Boersma Y L and PlütckthunA, 2011) are another class of proteins that mimic antibodies and canbind with high affinity and selectivity to target proteins. They arederived from the family of ankyrin proteins, which are adaptive proteinsthat bind integral membrane proteins to the spectral/actin network thatconstitutes the “spine” of the plasma cell membrane. The structure ofankyrins is based on the repetition of a motif of about 33 amino acidsand the same is true for DARPins. Each motif has a secondary structureof the helix-turn-helix type. DARPins contain at least three, preferablyfour to five repeated motifs and are obtained by screening combinatorialbanks.

The sensitization solution S1 includes the binding partner P1 in aconcentration that skilled persons will adapt according to thesensitivity of the test they wish to perform, the binding partner P1 tobe applied in the solid support, the analyte sought and the nature ofthe sample. Thus, for example, when the sensitization solution includesan antibody or antibody fragment, it may be used in a concentrationbetween 0.01 and 100 μg/mL. Unlike a conventional application process,in static or closed hollow support, the quantity of binding partner P1can be lower, for example by 10% compared to the concentration used instatic.

The sensitization solution S1 also includes all components necessary tofacilitate the application of the binding partner P1 in the solidsupport, such as pH buffers, for example carbonate, phosphate, TRIS,HEPES buffers, salts such as NaCl, preservatives such as azide, MIT;stabilizers such as glycerol, Tween 20, SDS, and saturation orpassivation proteins such as BSA, casein.

The container C1 containing the sensitization solution S1 can be anycontainer that contains a solution containing biological compounds suchas the binding partners P1. The only requirement is that the walls ofthe container are inert with respect to the binding partners P1 so thatthe latter do not attach themselves to the walls. Thus, the container C1can be made of plastic, such as ethylene vinyl acetate (EVA copolymer),polyethylene, polypropylene copolymer, fluorinated ethylene propylene(Nalgene), glass, or stainless steel. It can be in any form, forexample, vial, well, canister, box, jerry cans. This container C1 can beclosed before its implementation in the process of the invention andthen opened during the suction step. For example, this container C1 maybe a well covered with an aluminum foil cover that will be pierced bythe solid support itself when it draws up the sensitization solution.

Implemented with the same instrument means that the steps concerned usea single instrument, whereas implemented with two instruments of thesame range means that the steps concerned use two different instrumentsbut from the same manufacturer and with comparable features to beinterchangeable. The latter case refers indifferently to instruments ofthe same range or of the same type. Examples include the Applicant'sVIDAS® range, which includes the VIDAS®, mini VIDAS® and VIDAS® 3instruments. Thus, for example, the application of the bindingpartner(s) can be implemented with a VIDAS® instrument, then theanalysis can be implemented with another VIDAS® instrument or with aVIDAS® 3 instrument.

The first step in the process of the invention relates to the connectionof the solid support to a suction-discharge device. The connection canbe made manually or mechanically by the instrument in which the supportand the suction-discharge device are located.

The process of the invention includes several suction (ii)/contact(iii)/discharge (iv) cycles. The first step in this cycle is the suctionstep (ii) which consists in inserting the sensitization solution S1 intothe hollow solid support by one of its ends, without overflowing by theother end. It may be implemented by any method known to the skilledperson. For example, this suction step can be carried out by making apressure difference in the solid support, in particular by using apiston driven by any means known to the skilled person, such as a screwcoupled to a motor, forming a pump unit or by using a vacuum creationdevice. The pump unit can be at a varying distance from the solidsupport, for example through a tubing. Seals between the solid supportand the pump unit are present to seal it. All these elements are anexample of a suction/discharge device. The skilled person will adapt thesuction rate and power of the pump according to the amount ofsensitization solution to be introduced into the solid support and, ifnecessary, according to the length and cross-section of the tubingbetween the pump and the solid support. Thus, for example, in the caseof the VIDAS® instrument where the pump unit is close to the pipettetip, the suction speed will be between 10 and 180 μL/second. Asindicated above, the solid support can be used to directly draw up thesensitization solution from the container C1 or the suction can becarried out via a tubing on the one hand connected to the end of thesolid support by which the sensitization solution P1 is drawn up and onthe other hand immersed in the container C1.

Unlike the suction step (ii), the discharge step (iv) consists inremoving the sensitization solution S1 from the hollow solid support byone of its ends. The end through which the sensitization solution ispushed back can be the same as the one used to aspirate, as for examplein the case of the VIDAS® instrument, or it can be the opposite end.

The discharge container that collects the discharged sensitizationsolution can be the container C1, for example when the same end of thesolid support is used for suction and discharge. The discharge containermay also consist of a waste bin.

The discharge step can be carried out by any method known to the skilledperson, as described for the suction step. Conveniently, the samesystem, for example a pump unit as described above, is used for bothsuction and discharge steps.

Various other features emerge from the description given below withreference to the appended FIGS. 1 to 8, which show, by way ofnon-limiting examples, embodiments of the subject matters of theinvention.

FIG. 1 shows a diagram for the application of a binding partner, in asolid pipette tip-type support, with a loop coating system.

FIG. 2 shows a diagram for the application of a binding partner, in asolid pipette tip-type support, with an in-line system.

FIG. 3 shows a diagram for the application of a binding partner, in asolid pipette tip-type support, with a reciprocating system, asimplemented for example in the VIDAS® instrument.

FIG. 4 shows a strip and a VIDAS® pipette tip when implementing asuction cycle (FIG. 4A)/contact (FIG. 4B)/discharge (FIG. 4C) accordingto an embodiment of the invention for the application of a bindingpartner, the strip being made up of several containers including thecontainer S1 or S2.

FIG. 5 is a histogram-type representation of the relative fluorescencevalues (RFVs) for both sample types 1 and 2 when detecting TSH with theVIDAS® instrument, as a function of the different application conditionsof the binding partner (dynamically with a concentration of bindingpartner and a contact time that varies—2.5 μg/mL for 5, 10 or 15 min or5 μg/mL for 5, 10 or 15 min, or static with a concentration at 6 μg/mLfor 20 h).

FIG. 6 is a histogram-type representation of the relative fluorescencevalues (RFVs) for the three sample types (blank, low and high—FIG. 6A)or only for the blank sample (representation of the histograms in FIG.6A with enlarged scale—FIG. 6B) when detecting anti-Tg antibodies withthe VIDAS® instrument, as a function of the different applicationconditions of the binding partner (dynamic for 5, 15 or 30 min, orstatic for 20 h).

FIG. 7 is a graph showing the RFV signal as a function of TnIconcentration when detecting TnI with the VIDAS instrument (FIG. 7A: lowTnI concentration and FIG. 7B: high TnI concentration) using a pipettetip coated with 3 binding partners according to the process of theinvention (invention) or according to the prior art (reference), namelybiotinylated BSA, then streptavidin, then a mixture of biotinylatedanti-TnI antibodies.

FIG. 8 is a histogram giving the RFV signal for five samples withincreasing TSH concentration using either a pipette tip coated with ananti-TSH antibody according to the invention (dynamic coating at 4μg/mL) or a pipette tip coated with an anti-TSH antibody according tothe prior art (static coating at 6 μg/mL).

FIGS. 1 to 3 are particular embodiments of implementation ofsuction/contact/discharge cycles according to the process of theinvention.

According to FIG. 1, the device for applying the binding partner P1 inan open hollow support 1, of pipette tip type, comprises a container C14 containing the sensitization solution S1 containing said bindingpartner P1, a pump 2 which allows the sensitization solution tocirculate, via a tubing system 3, in the indicated arrow direction. Thefirst step consists of drawing up, in the direction of the arrow, usingpump 2, the sensitization solution from the container C1 4 into thesupport 1, then optionally leaving the sensitization solution and thesupport 1 in contact, then discharging, still in the direction of thearrows, using the pump 2, the sensitization solution from the support 1into the container 4, these steps being repeated. Thus, thesensitization solution is taken up at each step into the container 4,enters through one of the two ends of the support 1 and is dischargedinto the container 4 through the other end of the support 1.

According to a variant of this embodiment and as indicated above, thesensitization solution S1, after being drawn up into the support 1 byone of its ends, is discharged into the container C1 4 by the same end.

According to FIG. 2, the device for applying the binding partner P1 inan open hollow support 1, of pipette tip type, comprises a container C14 containing the sensitization solution S1 containing said bindingpartner P1, a pump 2 which allows the sensitization solution tocirculate, via a tubing system 3, in the indicated arrow direction. Thefirst step consists of drawing up, in the direction of the arrow, usingthe pump 2, the sensitization solution from the container C1 4 into thesupport 1, then optionally leaving the sensitization solution and thesupport 1 in contact, then discharging, still in the direction of thearrows, using the pump 2, the sensitization solution from the support 1into the container 5 which is a waste container, these steps beingrepeated and the sensitization solution being taken up at each step intothe container C1 4. Thus, the sensitization solution is taken up at eachstep into the container 4, enters through one of the two ends of thesupport 1 and is discharged into the container 5 through the other endof the support 1.

According to a variant of this embodiment, the suction/contact/dischargecycles are carried out using the same end of the support 1 and it isonly during the last cycle that the sensitization solution is dischargedinto a waste container.

According to FIG. 3, the device for applying the binding partner P1 inan open hollow support 1, of pipette tip type, comprises a container C14 containing the sensitization solution S1 containing said bindingpartner P1, a pump 2 which allows the sensitization solution tocirculate, via a tubing system 3, in the indicated arrow direction. Thefirst step consists of drawing up, in the direction of the arrow goingto the right, using the pump 2, the sensitization solution from thecontainer C1 4 into the support 1, then optionally leaving thesensitization solution and support 1 in contact, then discharging, stillin the direction of the arrow to the left, using the pump 2, thesensitization solution from the support 1 into the container 4, thesesteps being repeated. Thus, the sensitization solution is taken up ateach step into the container 4, enters through one of the two ends ofthe support 1 and is discharged into the container 4 from the same end.

An example of a process consisting of drawing up and discharging thesensitization solution using the same end of the hollow support is shownin FIG. 4 which represents a strip 6 and a VIDAS® pipette tip 1. Thetest strip 6 contains different wells, including a well 10 to receivethe test sample, a well 4 containing the sensitization solution S1, areading well 11 in which the fluorescence will be read after thebiological analysis of the sample is carried out, and wells 7, 8, 9which may contain different elements such as wash buffers or a wastewell in which the fraction of sensitization solution that comes intocontact with the pipette tip is discharged. FIG. 4A shows the first stepof the process of the invention, consisting in drawing up into thepipette tip 1, via a tubing 3 connected to a suction-discharge device(not shown in FIG. 4, but as shown in FIG. 3), a fraction of thesensitization solution contained in the well 4. FIG. 4B shows the secondstep during which the sensitization solution and the pipette tip 1 areleft in contact. Finally, FIG. 4C shows the discharge step consisting ofdischarging the sensitization solution into the well 4.

The contact step (iii) between the sensitization solution S1 and thesolid support is performed for a time between 0 s and 11 min. When thecontact time is 0 s, it means that the suction and discharge stepsfollow one another, without any pause time between the two. The contacttime can also be at most 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4min, 3 min, 2 min, 1 min, 55 s, 50 s, 45 s, 40 s, 35 s, 30 s, 25 s, 20 sor 15 s. The contact time can be at least 1 s, 2 s, 3 s, 4 s, 5 s, 6 s,7 s, 8 s, 9 s, 10 s, 11 s, 12 s, 13 s, 14 s, 15 s, 20 s, 30 s, 35 s or40 s.

The contact time also varies according to the number of cyclesimplemented, which is at least two. The minimum and maximum number ofcycles depends on the total duration of the application process,depending on the contact time chosen. Thus, the number of repetitioncycles of steps (i) to (ii) may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 18, 20 or 25 and at most 30, 35, 40, 45, 50, 55,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,120, 139, 150, 185, 200, 232, 250, 278, 300, 325, 371, 400, 417, 464,500, 510, 556, 600, 603, 649, 700, 800, 900, 1000, 2000, 2500, 3000,4000, 5000 or 6000 times.

The total time of the process is at least 1 min and at most about 2.5 h.This total time can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15 min and at most 2 h, 1.5 h, 1 h, 55 min, 50 min, 45 min, 40min, 39 min, 38 min, 37, min, 36 min, 35 min, 34 min, 33 min, 32 min, 31min, 30 min, 29 min, 28 min, 27, min, 26 min, 25 min, 24 min, 23 min, 22min, 21 min, 20 min, 19 min, 18 min, 17, min or 16 min.

The combination of contact time, number of cycles and total process timewill be chosen by skilled persons according to the sensitivity of theanalyte detection or quantification test they want to implement. Thiscombination will condition the amount of binding partner P1 that will beapplied to the inner surface of the solid support.

The temperature at which the application process of the invention iscarried out is any temperature compatible with the binding partner P1,i.e. at any temperature that maintains the binding capacities to theanalyte of said binding partner P1. In general, the temperature isbetween 10° C. and 45° C. Room temperature, from about 17° C. to about25° C., is therefore appropriate. The temperature can also be a functionof the instrument used. Thus, with regard to the VIDAS® instrument, thetemperature used is 37° C.

Once the binding partner P1 has been applied to the inner surface of thesolid support, it can be washed with wash solution W1 to remove unboundpartners. The wash solutions useful for this purpose are classic andknown to the skilled person. They include buffers, such as phosphate,Tris, HEPES buffers, salts such as NaCl, detergents such as 0.2% TritonX-100, Tween 20, etc. They may also contain saturation agents, alsocalled passivation agents, such as BSA or milk proteins, to saturate theinner surface of the solid support, thus avoiding non-specific bondsthat could occur when the test sample and the solid support arecontacted. Again, the only condition for this wash solution W1 is thatit must not destroy the binding capacity to the analyte of said bindingpartner P1.

The washing of the inner surface of the solid support may be carried outby any method known to the skilled person, such as soaking the solidsupport in a container containing said wash solution W1. It can also beimplemented by reproducing the suction (ii)/contact (iii)/discharge (iv)cycles as described above. Thus, according to an embodiment of theinvention, the application process also includes the following steps,implemented when the suction (ii)/contact (iii)/discharge (iv) cyclesare completed:

(v) draw up into the support in which the at least one binding partnerP1 is applied, a wash solution W1 contained in a container calledcontainer CW1, (vi) continue contact between the wash solution W1 andthe inner surface of the solid support for a time between 0 s and 11min,

(vii) discharge the wash solution W1 into a container, which isoptionally said container CW1,

steps (v) to (vii) forming a cycle that can be repeated at least once,over a total duration of at least 1 min and at most 2.5 h.

All the features described above for steps (ii), (iii) and (iv) alsoapply to steps (v), (vi) and (vii).

As indicated above, at the end of the discharge step, the solution usedcan be discharged into the wash container CW1 or into another containerwhich can then be the same container as the one used after step (iii).This would then be a waste container which will be identical both forthe application of the binding partner P1 and for the wash.

The binding partner P1 can be applied directly to the inner surface ofthe solid support. Or it can be applied through another binding partner,called binding partner P2 to binding partner P1 to the analyte. Hereagain, the concepts of analyte and binding partner P2 are quite distinctfrom each other. The analyte is the compound present in the test sample,while the binding partner P2 is the compound that will bind to thebinding partner P1 that will itself bind to the analyte. These bindingpartners P2 are optionally of immunological origin, as described above.When the binding partner P1 is an antibody, the binding partner P2 canbe an anti-species antibody, for example an anti-mouse antibody when thepartner P1 is a mouse antibody. The binding partner P2 can also be ananti-IgM antibody when the binding partner P1 is an IgM. When thebinding partner P1 is an antigen, for example a peptide, the bindingpartner P2 can be an anti-peptide antibody. The binding partner P1 mayhave been previously modified with a ligand, for example a biotin, thebinding partner P2 being an antiligand, for example streptavidin. Otherexamples of binding partners P2 are widely known to the skilled person.

The binding partner P2 can be contained in the sensitization solutionS1, which constitutes a particular embodiment of the invention. In thiscase, the binding partner P2/binding partner P1 formation on the solidsupport will be implemented during the suction (ii)/contact(iii)/discharge (iv) cycles of the application process of the invention.This is applicable when the partner P2 binds more easily to the supportthan the partner P1, for example when the partner P2 is streptavidin andthe partner P1 is a biotinylated antibody.

The binding partner P2 may also have previously been applied to theinner surface of the solid support according to any process known to theskilled person, for example in static or dynamic mode, reproducing thesame cycles as those described above for the application of the bindingpartner P1.

Thus, at least one binding partner P2 to a binding partner P1 to ananalyte to be detected or quantified in a test sample the bindingpartner P2 can be applied in the tubular solid support, optionallyflared, having a circular or ellipsoidal opening at each end, such as apipette tip or a pipette, by a method comprising the following steps:(a) connect the solid support to a suction-discharge device,

(b) draw up into the solid support, by one of its ends, a solutioncomprising said at least one binding partner P2, called sensitizationsolution S2, contained in a container, called container C2,

(c) continue contact between the sensitization solution S2 and the innersurface of the solid support for a time between 0 s and 11 min,

(d) discharge the sensitization solution S2 into a container, which isoptionally said container C2,

steps (b) to (d) forming a cycle that can be repeated at least once,over a total duration of at least 1 min and at most 2.5 h.

All the features described above for steps (ii), (iii) and (iv) alsoapply to steps (b), (c) and (d).

As indicated above, at the end of the discharge step, the solution usedcan be discharged into the sensitization container C2 or into anothercontainer which can then be the same container as the one used with theother discharge steps previously described (waste container).

After application of the binding partner P2 to the inner surface of thesolid substrate, it can be washed with a wash solution W2 to removeunbound partners.

Washing may also include the following steps, implemented when thesuction (b)/contact (c)/discharge (d) cycles are completed:

(e) draw up into the support in which the at least one binding partnerP2 is applied, a wash solution W2 contained in a container calledcontainer CW2,

(f) continue contact between the wash solution W2 and the inner surfaceof the solid substrate for a time of between 0 s and 11 min,

(g) discharge the wash solution W2 into a container, which is optionallysaid container CW2,

steps (e) to (g) forming a cycle that can be repeated at least once,over a total duration of at least 1 min and at most 2.5 h.

The wash solution W2 has the same features as the wash solution W1described above. It may be identical (in which case it will be referredto indifferently as wash solution W for solution W1 and solution W2) orit may be different from wash solution W1, both in terms of nature andin terms of its container. It is preferably in a different container CW2from CW1, but washing can be carried out with the same solutioncontained in the same container (this is called container CW).

As before, the discharge of the wash solution can be carried out in thewash container itself (CW2 or CW) or in a waste container, for examplecommon for all discharge steps.

Whether or not there are washing steps, in order to use the solidsupport to detect or quantify an analyte likely to be contained in atest sample, it will then be coated with the binding partner P1.According to an embodiment, the application process of the bindingpartner P2 also includes the application, in said solid support, afterthe suction (b)/contact (c)/discharge (d) cycles, of said at least onebinding partner P1.

The binding partner P1 can be applied to the inner surface of the solidsupport according to any process known to the skilled person, forexample in static or dynamic mode, such that the same cycles of steps(ii) to (iv) described above can be reproduced for the application ofsaid binding partner P1, which is another embodiment.

The application process of the binding partner P1 can be delayedrelative to the application process of the binding partner P2, i.e.there is a waiting time of at least 5 min between the two processes, orit can be implemented immediately after the application process of thebinding partner P2. Preferably, they should be used with the sameinstrument or with two instruments from the same range.

The different containers used in the application process of the bindingpartners can be physically separated, handled independently from oneanother, such as vials that are placed in a carousel of an instrument.Or the containers may be integral, for example in the form of a well ina strip, such as the VIDAS® strip (bioMérieux) shown in FIG. 4 (strip6). According to an embodiment, the container C1, the container CW1, andif necessary the container C2 and/or the container CW2 are contained inthe same test strip, which consists of several containers.

Since the test strip or instrument incorporating the differentcontainers is intended for the in vitro detection or quantification ofan analyte, it may also include other containers containing othercomponents necessary for the detection or quantification of saidanalyte. The components necessary for the detection or quantification ofsaid analyte are known to the skilled person and will be described belowin the context of the detection or quantification of the analyte.Examples of such components include labelled binding partners, alsoknown as conjugates, wash solutions and detection substrates. Examplesof containers include a VIDAS® strip with all reagents (for coating anddetection or quantification), or two VIDAS® strips, one for coating andthe other for detection or quantification.

The in vitro detection or quantification of an analyte likely to becontained in a test sample may be carried out by any in vitro processknown to the skilled person using at least one binding partner P1 to theanalyte. It comprises the steps of contacting the test sample with thesolid support coated with the binding partner P1 and detecting thebinding, if the analyte is present, of said analyte and said at leastone binding partner P1.

The application process of the binding partner P1 has been describedabove. The support can also include a binding partner P2, also appliedas described above.

The analyte and the test sample that may contain it are also asdescribed above. According to one embodiment, the test sample is asample of biological, chemical, food or environmental origin.

The detection or quantification of analyte may be carried out by anyanalytical method known to the skilled person, such as immunoassaymethods. These so-called enzyme-linked immunoassay (EIA) methods arecoupled to an enzyme-catalyzed reaction using an enzyme substrate.Depending on the enzyme substrate chosen, a colorimetric signal(enzyme-linked immunosorbent assay, ELISA) (Rassasie, M. J., et al.,1992), a fluorescence signal (enzyme-linked fluorescent assay, ELFA) ora chemiluminescent signal (chemiluminescence immunoassay, CLIA) (StablerT. V., et al., 1991) can be produced.

These methods are based on measurements to quantify the signals emittedduring the analysis of the test sample. The quantity of signals detectedis generally proportional to the amount of analyte to be measured (forexample in a sandwich test) or inversely proportional to the amount ofanalyte to be measured (for example in a competition test).

Conventional steps in the in vitro detection and/or quantificationprocess of an analyte by sandwich immunoassay, in a test sample likelyto contain said analyte, include or consist of:

-   -   the presence of the solid support inside which is applied said        at least one binding partner P1 and said sample for fixing the        analyte on the partner P1,    -   the addition of a detection partner, which is directly or        indirectly coupled to a label, such as an enzyme capable of        lysing an enzymatic substrate, for example fluorogen for ELFA        detection, for its binding to the binding partner P1-analyte        complex,    -   when the label is an enzyme, contacting an enzyme substrate and        the binding partner P1-analyte-detection partner complex coupled        to an enzyme to form a reaction medium, and    -   detection, for example by immunofluorescence in ELFA detection,        of the presence and/or amount of analyte by measuring the signal        (for example fluorescence) emitted in the reaction medium.

Detection partner means any partner capable of binding to the analyte tobe detected or quantified, which will be coupled directly or indirectlyto a label, for example an enzyme. It may be of the same nature as thebinding partner P1 or of a different nature. Examples are given abovewith the binding partner P1.

Direct or indirect coupling of the label to the detection partner meansthat the label is attached directly to the detection partner recognizingthe analyte (direct coupling) or the enzyme is coupled to a bindingpartner that recognizes the detection partner which recognizes theanalyte itself (indirect coupling).

Thus, in the context of direct coupling, the complex formed at the endof the assay, called a conjugate, will consist of: “Capturepartner/analyte/detection partner coupled to the label”.

As part of the indirect coupling, the complex formed at the end of theassay will consist of: “Capture partner/analyte/detectionpartner/binding partner coupled to the label”.

In the latter case, the binding partner is well known to the skilledperson and may be, for example, an anti-IgG (immunoglobulin) antibodywhen the detection partner is an IgG recognizing the analyte ofinterest.

Label means in particular any molecule containing a reactive group witha group of the detection partner, directly without chemicalmodification, or after chemical modification to include such a group,which molecule is capable of generating a detectable signal directly orindirectly. A non-exhaustive list of these direct detection markersconsists of:

enzymes that produce a detectable signal for example by colorimetry,fluorescence, luminescence, such as horseradish peroxidase, alkalinephosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase,

chromophores such as fluorescent compounds, luminescent compounds, dyes,

radioactive molecules such as 32P, 35S or 125I,

fluorescent molecules such as Alexa or phycocyanins, and

electrochemiluminescent salts such as organometallic derivatives basedon acridinium or ruthenium.

According to a particular embodiment, the binding partner P1 used in thedetection or quantification process is an immunoassay partner and thedetection of the binding of said analyte is implemented by a sandwichtest using another binding partner to the analyte, called the detectionpartner, optionally of a different nature, which is labelled by a label.

Conventional steps in the process of in vitro detection and/orquantification of an analyte by competitive immunoassay in a test samplelikely to contain said analyte include:

-   -   the presence of the solid support inside which is applied said        at least one binding partner P1, an analyte analogue coupled to        a label, for example an enzyme capable of lysing an enzymatic        substrate, for example fluorogen, and said sample, which compete        for binding to the binding partner P1,    -   when the label is an enzyme, contacting an enzyme substrate,        binding partner P1-analyte and binding partner P1-analogue to        the analyte complexes to form a reaction medium, and    -   the detection, for example by immunofluorescence, of the        presence and/or amount of analyte by measuring the signal, for        example fluorescence, emitted in the reaction medium.

Analogue to the analyte means any molecule that has the same bindingcapabilities to the binding partner P1 as the analyte.

The label coupled to the analogue to the analyte is equivalent to thelabel used in a sandwich test.

According to another particular embodiment of the invention, the bindingpartner P1 used in the detection or quantification process is animmunoassay partner and the detection of the binding or not of saidanalyte is implemented by a competitive test using a labelled compoundcompeting with the analyte to be detected or quantified, otherwisecalled the labelled analogue to the analyte.

Regardless of the type of method used, whether in sandwiches or incompetition, the enzyme is a widely appropriate label and examplesinclude sulfatase, alkaline phosphatase (ALP), acid phosphatase, glucoseoxidase (GOx), glucose-6-phosphate dehydrogenase (G6PD) andβ-galactosidase (β-gal). The corresponding enzyme substrates are widelyknown to the skilled person and include, for example,4-methylumbelliferyl phosphate or5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside.

According to a particular embodiment, in the detection or quantificationprocess according to the invention, the detection of the analyte iscarried out using an enzyme and an enzymatic substrate catalyzed by saidenzyme, preferably alkaline phosphatase and 4-methylumbelliferylphosphate.

The analyte detection or quantification process may also include one ormore additional washing steps after each step, such as:

-   -   before the addition of the detection partner, a washing step to        remove the analyte not bound to the binding partner P1-analyte        complex; and    -   after the addition of the detection partner, a washing step to        eliminate the unbound detection partner.

The washing steps are steps known to the skilled person. They areimplemented with buffers compatible with the reaction medium and signalreading.

The method of detection or quantification of an analyte can beimplemented at any time after the application process of the bindingpartner P1 to the inner surface of the solid support, and if applicablethe binding partner P2. It can be implemented one or more days after theapplication of the binding partner P1 and, if necessary, the bindingpartner P2, or even one or more weeks later. In this case, the solidsupport must be dried and then stored in a desiccant bag to avoid anystability problems. Advantageously, the steps of contacting the testsample with the solid support and detecting the binding, if the analyteis present, of said analyte and said at least one binding partner P1 ofthe detection or quantification process of the invention are carried outimmediately after the process of applying at least one binding partnerP1 in said solid support as defined above. Immediately after means thatthe detection or quantification process is implemented within minutes oreven seconds of the application process of the binding partner P1. Thereare no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minbetween the last step of the binding partner P1 application process andthe first step of the analyte detection or quantification process. Thisshort time may be useful to introduce the test sample and/or containersthat will be used for detection or quantification. In other words, theapplication process of at least one binding partner P1, or even at leastone partner P2 beforehand, and the detection or quantification processare implemented successively.

In order to facilitate the user's organization, to control the costs andspace of the laboratory, the analyte detection or quantification processis implemented with the same instrument or type of instrument as thatused for the application of the binding partner P1, or even the bindingpartner P2, or even the different washing processes. Also, the steps ofcontacting the test sample with the solid support on which said at leastone binding partner P1 is applied, and detecting the binding of saidanalyte and said at least one binding partner P1, include or consist ofthe following steps consisting in:

(1) drawing up into said solid support, by one of its ends, said samplecontained in a container, called container CE, leaving in contact anddischarging said sample into a container, which is optionally saidcontainer CE,(2) drawing up into said solid support, at the same end, a solutioncomprising a compound conjugated to a label, such as another bindingpartner to the analyte or a compound competing with the analyte, calledconjugate solution CS, said conjugate solution C being contained in acontainer called container CC, leaving in contact and discharging saidconjugate solution CS into a container, which is optionally saidcontainer CC,(3) if the label uses a detection substrate, drawing up into said solidsupport, at the same end, a solution comprising a detection substratewith which the label will react, called substrate solution SS, saidsubstrate solution SS being contained in a container called containerCS, leaving in contact and discharging said substrate solution into acontainer, which is optionally said container CS, and(4) measuring the transmitted signal.

If necessary, these steps are preceded by a step of connecting the solidsupport to a suction-discharge device such as a pump unit.

Steps (1) and (2) of suction and discharge can be implemented asdescribed above.

The different components used, for example the binding partners P1, thedetection partners, markers, etc. are as described above.

The different containers CE, CC and CS used are as described above andcan be separated or contained in the same test strip, for example, whichis a particular embodiment.

The last step (4) consists in measuring the transmitted signal. Thisstep is well known to the skilled person. This measurement can betransformed into a representative amount by the user such as theconcentration of analyte using a standard curve, also called acalibration curve, the obtaining of which is widely known to the skilledperson and can be obtained by i) measuring the signal generated bystandards, also called standards or calibrators, then ii) plotting thecurve giving the signal as a function of amount or concentration. Veryoften, it is common practice to find a mathematical model thatrepresents, as accurately as possible, this relationship between signaland amount or concentration, in order to be able to easily calculate theresults of a quantitative immunoassay.

In addition to steps (1) to (4), the process may also include washingsteps, as described above.

The invention will be better understood using the following examples,which are given by way of non-limiting illustration.

EXAMPLES Example 1: Application of an Anti-TSH Antibody in a Pipette Tipand Detection of TSH

Thyrotropin-releasing hormone or thyroid stimulating hormone (TSH) is ahormone secreted by the thyroid cells of the anterior pituitary gland.This hormone is the main stimulating factor of the thyroid gland thatdetermines the production of the thyroid hormones T3 and T4. In return,these thyroid hormones exert a feedback control on the anteriorpituitary gland, which slows down the secretion of TSH. The secretion ofTSH is also controlled by the central nervous system through ahypothalamic neuropeptide, TRH, and neurotransmitters such assomatostatin or dopamine. The blood test for TSH is an aid in thediagnosis of thyroid or pituitary disorders.

1.1 Application of an Anti-TSH Antibody in a VIDAS® Pipette TipAccording to the Invention

VIDAS® pipette tips are sensitized with 300 μL of a solution of mousemonoclonal anti-TSH antibody (bioMérieux product number 30400) at 2.5 or5 μg/mL in a Tris HCl buffer pH 7.3 (sensitization solution) containedin one of the wells of a VIDAS® strip, using the VIDAS® instrument, asfollows:

Five-Minute Protocol:

-   -   100 μL/s aspiration of the sensitization solution    -   incubation 10 seconds    -   solution discharge.

This cycle is repeated 20 times.

Ten-Minute Protocol:

-   -   100 μL/s aspiration of the sensitization solution    -   incubation 18 seconds    -   solution discharge.

This cycle is repeated 25 times.

Fifteen-Minute Protocol:

-   -   100 μL/s aspiration of the sensitization solution    -   incubation 20 seconds    -   solution discharge.

This cycle is repeated 35 times.

The emptied pipette tips are ready to be used with no furtherpreparation for the determination of TSH in a biological sample.

1.2 Application of an Anti-TSH Antibody in a VIDAS® Pipette TipAccording to the Prior Art

For comparison purposes, the VIDAS® pipette tips were sensitizedstatically as follows: The pipette tips are sensitized with 300 μL ofthe same mouse anti-TSH monoclonal antibody solution except that theantibody concentration is 6 μg/mL. After about 20 hours at roomtemperature (18-25° C.) with the sensitization solution, the pipettetips are emptied. Then, 330 μL of a saturation solution containing inparticular animal proteins are added for passivation of the pipette tipsfor about 6 hours. The pipette tips are then emptied, dried and storedat 4° C. until use, away from moisture.

1.3 Determination of TSH in a Sample

The pipette tips coated with anti-TSH antibodies according to points1.1. and 1.2. above are used with strips from the VIDAS® TSH Kit(bioMérieux product number 30400), which include the other reagents ofthe immunological reaction.

All assay steps are performed automatically by the VIDAS® instrumentaccording to a standard procedure for this instrument.

The sample contained in the sample well (200 μL) of the strip iscollected and transferred to the well containing the alkalinephosphatase (conjugate) labelled anti-TSH antibody. The sample/conjugatemixture is drawn up into and then successively discharged by the pipettetip for about 16 minutes. This operation allows the antigen to bind tothe immunoglobulins attached to the pipette tip and to the conjugateforming a “sandwich”.

Three successive washing steps of 3 cycles of 5 seconds each, carriedout by the instrument using the wash solution contained in the VIDAS TSHKit strip, remove the unbound compounds.

In the final detection step, the substrate (4-methylumbelliferylphosphate) contained in a well of the strip is drawn up into the pipettetip and then discharged into the detection well; the enzyme of theconjugate catalyzes the hydrolysis reaction of this substrate into aproduct (4-methylumbelliferone) whose emitted fluorescence is measuredat 450 nm in the detection well. The value of the fluorescence signal(RFV=relative fluorescence value) is proportional to the concentrationof the antigen present in the sample.

Two types of human serum samples containing TSH were used for eachconcentration condition and protocol duration, namely a sample 1containing a normal TSH concentration (3 μIU/mL TSH) and a sample 2containing a high TSH concentration, corresponding to hypothyroidism (40μIU/mL TSH).

The results are given in FIG. 5 which is a histogram-type representationof the relative fluorescence values (RFVs) for both types of sample 1and 2 when detecting TSH with the VIDAS® instrument, as a function ofthe different application conditions of the binding partner (dynamicallywith a binding partner concentration and contact time that vary—2.5μg/mL for 5, 10 or 15 min or 5 μg/mL for 5, 10 or 15 min, or static witha concentration at 6 μg/mL for 20 h).

FIG. 5 shows that the RFV signal obtained for sensitizationconcentrations of 2.5 μg/mL and 5 μg/mL in dynamic mode is quitecomparable, regardless of the duration of sensitization. From 5 min ofdynamic coating, the RFV signal for sample 1 is about 500 RFV. Sample 2yields RFVs greater than 3500. All the dynamic application conditions ofthe anti-TSH antibody used according to the invention have excellentsignal dynamics, equivalent to the reference condition obtained inseveral days by a static incubation process.

Example 2: Application of a Tg Antigen in a Pipette Tip and Detection ofAnti-Tg Antibodies

Thyroglobulin (Tg) is a glycoprotein produced in the thyroid gland andis the main component of follicular colloid. Its main role is thestorage and synthesis of thyroid hormones. Antithyroglobulinautoantibodies are often present in patients with autoimmune thyroiddisease. For example, they are detected in 30% of patients with Graves'disease (Basedow) and in 85% of patients with Hashimoto's disease (2).Anti-Tg antibodies are associated with hypothyroidism or mildhyperthyroidism and are frequently present in patients with otherautoimmune diseases such as rheumatoid arthritis, pernicious anemia andtype I diabetes (3, 4).

2.1. Application of a Tg Antigen in a VIDAS® Pipette Tip According tothe Invention

VIDAS® pipette tips are sensitized with 300 μL of a native Tg antigensolution (bioMérieux product number 30462) at 7 μg/mL in a phosphatebuffer (sensitization solution) contained in one of the wells of aVIDAS® strip, using the VIDAS® instrument, as follows:

Five-Minute Protocol:

-   -   100 μL/s aspiration of the sensitization solution    -   incubation 10 seconds    -   solution discharge.

This cycle is repeated 20 times.

Fifteen-Minute Protocol:

-   -   100 μL/s aspiration of the sensitization solution    -   incubation 20 seconds    -   solution discharge.

This cycle is repeated 35 times.

Thirty-Minute Protocol:

-   -   100 μL/s aspiration of the sensitization solution    -   incubation 30 seconds    -   solution discharge.

This cycle is repeated 50 times.

The emptied pipette tips are ready to be used with no furtherpreparation for the determination of anti-Tg antibodies in a biologicalsample.

2.2 Application of a Tg Antigen in a VIDAS® Pipette Tip According to thePrior Art

For comparative purposes, the VIDAS® pipette tips were sensitizedstatically as follows:

The pipette tips are sensitized with 300 μL of the same native Tgantigen solution. After about 6 hours at room temperature (18-25° C.)with the sensitization solution, the pipette tips were emptied. Then,330 μL of a saturation solution containing in particular animal proteinsare added for passivation of the pipette tips for about 6 hours. Thepipette tips are then emptied, dried and stored at 4° C. until use, awayfrom moisture.

2.3 Determination of Anti-Tg Antibodies in a Sample

The pipette tips thus coated with Tg antigen according to points 2.1.and 2.2. above are used with strips from the VIDAS anti-TG kit (productnumber 30462).

The sample (100 μL) is taken by the instrument from the sample well ofthe strip and then transferred to the well containing a sample diluent.The diluted sample is aspirated and then discharged for about 3 minutes.This step allows the anti-Tg antibodies present in the sample to bind tothe antigen attached to the pipette tip. The unbound components of theserum are removed by 3 washes in wells of the VIDAS anti-Tg strip forabout 3 minutes. An incubation step with the detection conjugate isperformed for about 6 minutes, respecting suction/discharge cycles of30×8 seconds. The conjugate binds specifically to the anti-Tg antibodiesof the previously bound sample. A washing cycle identical to theprevious one removes the excess of unbound conjugate before thedetection.

In the final detection step, the substrate (4-methylumbelliferylphosphate) contained in a well of the strip is drawn up into the pipettetip and then discharged into the detection well; the enzyme of theconjugate catalyzes the hydrolysis reaction of this substrate into aproduct (4-methylumbelliferone) whose emitted fluorescence is measuredat 450 nm in the detection well. The value of the fluorescence signal(RFV=relative fluorescence value) is proportional to the concentrationof the antigen present in the sample.

The assayed samples are samples of natural human serum with aconcentration corresponding to 60 IU/mL (low sample) and 1000 IU/mL(high sample). The blank sample is a mixture of negative samples.

The results are given in FIG. 6 which is a histogram-type representationof the relative fluorescence values (RFVs) for the three sample types(blank, low and high—FIG. 6A) or only for the blank sample(representation of histograms in FIG. 6A with enlarged scale—FIG. 6B)when detecting anti-Tg antibodies with the VIDAS® instrument, as afunction of the different application conditions of the binding partner(dynamically for 5, 15 or 30 minutes, or statically for about 6 hours).

FIG. 6A shows that the dynamic application conditions for binding the Tgantigen to the support according to the process of the invention givesignals equivalent to or better than those obtained with the staticreference condition. The signal obtained on low and high human serumsamples is satisfactory after 5 minutes of dynamic adsorption time. Itis at its maximum for the 30-minute protocol, allowing a higher signalthan the reference with the high sample.

The signal of the blank sample is used to evaluate the non-specificsignal. The signal for this sample must be at the lowest level. As shownin FIG. 6B, with dynamic application conditions, this non-specificsignal is weaker (less than 20 RFV) than for the static reference forwhich the signal is 25 RFV.

The conditions of application of the Tg antigen on the support accordingto the invention lead to an immunoassay equivalent to or better than thereference condition.

Example 3: Application of Several Binding Partners to Form aBiotinylated BSA/Streptavidin/Biotinylated Anti-cTni Antibody Complexand Detection of TNI

Troponin is a protein complex that sensitizes muscle cells to thecalcium responsible for inhibiting the binding between myosin and actin(by masking the actin site that is used for binding to myosin). Ittherefore has an inhibitory function that has the effect of initiatingmuscle relaxation. protein used. Troponin I (TnI) is a subunit. Itsdetermination is widely used as a tool to assist in the diagnosis ofmyocardial infarction (MI) and 30-day risk stratification for all-causemortality and major adverse cardiac events (MACE) including myocardialinfarction and revascularization in patients with acute coronarysyndrome (ACS)-like symptoms.

3.1 Dynamic Sensitization of Pipette Tips with Biotinylated BSA andStreptavidin for the Binding of Specific Biotinylated Anti-cTniAntibodies

VIDAS® pipette tips are sensitized with 300 μL of a biotinylated BSAsolution (bioMérieux product number 30448) at 1 μg/mL in a carbonatebuffer contained in one of the wells of a VIDAS® strip. The cyclesperformed by the VIDAS® and repeated 50 times are:

-   -   100 μL/s aspiration of the biotinylated BSA solution,    -   incubation for 20 s then    -   solution discharge.

Next, these same pipette tips are incubated with 300 μL of a 5 μg/mLstreptavidin solution (bioMérieux product number 30448) diluted in PBSbuffer for 50 cycles of 20 seconds (aspiration, incubation of 20 s anddischarge).

The pipette tips are then sensitized with a mixture of biotinylatedanti-cTnI antibodies (bioMérieux product number 30448) at 2 μg/mL and 3μg/mL respectively, on 300 μL. The incubation time is 50 times 20seconds.

After each of these 3 incubation steps, the pipette tips are washedtwice successively on 300 μL for 8 times 1 second in wells containing awash solution in the VIDAS cartridge. The emptied pipette tips are readyto be used with no further preparation for the determination of TnI in abiological sample.

3.2. Application of Biotinylated BSA and then Streptavidin for theBinding of Specific Biotinylated Anti-cTni Antibodies in a VIDAS®Pipette Tip According to the Prior Art

For comparative purposes, the VIDAS® pipette tips were sensitizedstatically as follows:

The pipette tips are sensitized with 300 μL of the same biotinylated BSAsolution for about 20 hours at room temperature (18-25° C.). The pipettetips are then emptied and filled with 300 μL of streptavidin solutionfor about 20 hours at room temperature (18-25° C.). After emptying thepipette tips, the sensitization step on approximately 300 with themixture of biotinylated anti-cTni antibodies at the same concentrationas in point 3.1. continues for approximately 20 hours. The pipette tipsare then emptied, dried and stored at 4° C. until use, away frommoisture.

3.2. Determination of cTnI

All test steps are performed automatically by the instrument accordingto a standard instrument operating mode. They consist of a succession ofsuction/discharge cycles of the reaction medium. The sample (200 μL) iscollected and transferred to the well containing the alkalinephosphatase (conjugate) labelled cardiac anti-troponin antibodies. Thesample/conjugate mixture is drawn up into and then successivelydischarged by the pipette tip for about 10 minutes (50×8 seconds). Thisoperation allows the antigen to bind to the immunoglobulins attached tothe pipette tip and to the conjugate forming a “sandwich”.

Three successive washing steps of 3 cycles of 2 seconds each removeunbound compounds.

Two detection steps are then carried out successively. At each step, thesubstrate (4-methylumbelliferyl phosphate) is drawn up into and thendischarged into the pipette tip; the enzyme of the conjugate catalyzesthe hydrolysis reaction of this substrate into a product(4-methylumbelliferone) whose emitted fluorescence is measured at 450nm. The value of the fluorescence signal is proportional to theconcentration of the antigen present in the sample.

At the end of the test, the results are automatically calculated by theinstrument against two stored calibration curves corresponding to thetwo detection steps. A threshold signal controls the choice of thecalibration curve to be used for each sample. Then the results areprinted.

The assayed samples are human sera with Tni concentrations ranging from0.001 μg/L to 14.03 μg/L (samples 1 to 11).

RFV signal results as a function of TnI concentration are given in FIG.7 (FIG. 7A: samples 1 to 6 and FIG. 7B: samples 7 to 11) which showsthat the signal is equivalent between the two types of coating,regardless of the concentration of the sample tested. The application ofthe biotinylated BSA/streptavidin/biotinylated antibody complexaccording to the invention makes it possible to obtain an efficientimmunoassay, according to a much faster process than with the prior art.

Example 4: Variation of Binding Partner Concentrations in theSensitization Solution

4.1. Application of an Anti-TSH Antibody in a VIDAS® Pipette TipAccording to the Invention

VIDAS® pipette tips are sensitized with 300 μL of a 4 μg/mL solution ofmouse monoclonal anti-TSH antibody in a Tris HCl buffer pH 7.3(sensitization solution) contained in one of the wells of a VIDAS®strip, using the VIDAS® instrument, as follows:

Sixty-Minute Protocol:

-   -   100 μL/s aspiration of the sensitization solution    -   incubation 20 seconds    -   solution discharge.

This cycle is repeated 139 times.

The empty pipette tips are ready for use with no further preparation forthe determination of TSH in biological samples.

4.2. Application of an Anti-TSH Antibody in a VIDAS® Pipette TipAccording to the Prior Art

For comparative purposes, the VIDAS® pipette tips are sensitizedaccording to the prior art as follows:

The pipette tips are sensitized with 300 μL of a solution of mousemonoclonal anti-TSH antibody (bioMérieux product number 30400) at 6μg/mL in a Tris HCl pH 7.3 buffer. After about 20 hours at roomtemperature (18-25° C.) with the sensitization solution, the pipettetips are emptied. Then, 330 μL of the same solution containing animalproteins are added for passivation of the pipette tips for about 6hours. The pipette tips are then emptied, dried and stored at 4° C.until use, away from moisture.

4.3. Determination of TSH

The TSH assay is implemented as described in Example 1. The assayedsamples (SC13, SC14, SC15, SC16 and SC17) are human sera with increasingTSH concentrations.

The results of these assays are given in FIG. 8 which is ahistogram-type representation giving the RFV signal for each sampleusing either a pipette tip coated with an anti-TSH antibody according tothe invention (dynamic coating at 4 μg/mL) or a pipette tip coated withan anti-TSH antibody according to the prior art (static coating at 6μg/mL).

FIG. 8 shows that the signals obtained between the two pipette tipfabrications (according to the invention and according to the prior art)are very comparable, whereas the fabrication of the invention uses ⅓less anti-TSH antibodies in the sensitization solution (4 μg/mL versus 6μg/mL) and allows a production of these pipette tips for use inimmunoassay in 1 hour versus 2 steps of more than 6 hours each accordingto the prior art. All other experimental conditions being identical, theapplication of anti-TSH antibodies according to the invention allowssubstantial savings in raw materials and time.

REFERENCES

-   Boersma Y L and Plütckthun A, 2011, Curr. Opin. Biotechnol,    22:849-857-   Ellington A D and Szostak J W., 1990, Nature, 346:818-822-   Rassasie M. J. et al., 1992, Steroids, 57:112-   Stabler T. V., et al., 1991, Clin. Chem., 37(11):1987

1. A process for applying, in a tubular solid support, optionallyflared, having a circular or ellipsoidal opening at each end, at leastone binding partner P1 to an analyte to be detected or quantified in atest sample, comprising the following steps: (i) connect the solidsupport to a suction-discharge device, (ii) draw up into the support, byone of its ends, a solution comprising the at least one binding partnerP1, called sensitization solution S1, contained in a container, calledcontainer C1, (iii) continue contact between the sensitization solutionS1 and the inner surface of the solid support for a time between 0 s and11 min, (iv) discharge the sensitization solution S1 into a container,which is optionally the container C1, steps (ii) to (iv) forming a cyclethat can be repeated at least once, over a total duration of at least 1min and at most 2.5 h.
 2. The application process as claimed in claim 1,wherein the contact time of step (iii) between the sensitizationsolution S1 and the inner surface of the solid support is between 2 sand 1 min.
 3. The application process as claimed in claim 1, whereinsteps (ii) to (iv) are repeated from 10 to 100 times.
 4. The applicationprocess as claimed in claim 1, wherein the total of the repeated cyclesis between 10 and 20 min.
 5. The application process as claimed in claim1, wherein it also includes the following steps, carried out when thesuction (ii)/contact (iii)/discharge (iv) cycles are completed: (v) drawup into the solid support in which the at least one binding partner P1is applied, a wash solution W1 contained in a container called containerCW1, (vi) continue contact between the wash solution W1 and the innersurface of the solid support for a time between 0 s and 11 min, (vii)discharge the wash solution W1 into a container, which is optionally thecontainer CW1, steps (v) to (vii) forming a cycle that can be repeatedat least once, over a total duration of at least 1 min and at most 2.5h.
 6. The application process as claimed in claim 1, wherein thesensitization solution S1 also contains at least one binding partner P2to the binding partner P1 to the analyte.
 7. A process for applying, ina tubular solid support, optionally flared, having a circular orellipsoidal opening at each end, at least one binding partner P2 to abinding partner P1 to an analyte to be detected or quantified in a testsample, comprising the following steps: (a) connect the solid support toa suction-discharge device, (b) draw up into the solid support, by oneof its ends, a solution comprising the at least one binding partner P2,called sensitization solution S2, contained in a container, calledcontainer C2, (c) continue contact between the sensitization solution S2and the inner surface of the solid support for a time between 0 s and 11min, (d) discharge the sensitization solution S2 into a container, whichis optionally the container C2, steps (b) to (d) forming a cycle thatcan be repeated at least once, over a total duration of at least 1 minand at most 2.5 h.
 8. The application process as claimed in claim 7,wherein it also includes the following steps, carried out when thesuction (b)/contact (c)/discharge (d) cycles are completed: (e) draw upinto the support in which the at least one binding partner P2 isapplied, a wash solution W2 contained in a container called containerCW2, (f) continue contact between the wash solution W2 and the innersurface of the solid substrate for a time between 0 s and 11 min, (g)discharge the wash solution W2 into a container, which is optionally thecontainer CW2, steps (e) to (g) forming a cycle that can be repeated atleast once, over a total duration of at least 1 min and at most 2.5 h.9. The application process as claimed in claim 7, also comprising theapplication, in the solid support, after the suction (b)/contact(c)/discharge (d) cycles, of the at least one binding partner P1. 10.(canceled)
 11. The application process as claimed in claim 5, whereinthe container C1, the container CW1, and optionally the container C2and/or the container CW2 are contained in the same test strip, whichconsists of several containers.
 12. The application process as claimedin claim 11, wherein the test strip also includes other containerscontaining other components necessary for the detection orquantification of the analyte.
 13. A process for the in vitro detectionor quantification of an analyte in a test sample likely to contain theanalyte, the process using at least one tubular solid support,optionally flared, having a circular or ellipsoidal opening at each end,in which at least one binding partner P1 to an analyte to be detected orquantified is applied in the test sample according to an applicationprocess as defined in claim 1, which detection or quantification processcomprises the steps of contacting the test sample with the solid supportand detecting the binding, if the analyte is present, of the analyte andthe at least one binding partner P1.
 14. The detection or quantificationprocess as claimed in claim 13, wherein the binding partner P1 is animmunoassay partner and the detection of the binding of the analyte iscarried out by a sandwich test using another binding partner to theanalyte, optionally of a different nature, which is labelled.
 15. Thedetection or quantification process as claimed in claim 13, wherein thebinding partner P1 to the analyte is an immunoassay partner and thedetection or not of the binding of the analyte is carried out by acompetitive test using a labelled compound competing with the analyte tobe detected or quantified.
 16. The detection or quantification processas claimed in claim 13, wherein the detection of the analyte is carriedout using an enzyme and an enzymatic substrate catalyzed by the enzyme.17. (canceled)
 18. The detection or quantification process as claimed inclaim 13, wherein the steps of contacting the test sample with the solidsupport on which the at least one binding partner P1 is applied, anddetecting the binding of the analyte and the at least one bindingpartner P1, include the following steps consisting in: (1) drawing upinto said solid support, by one of its ends, the sample contained in acontainer, called container CE, leaving it in contact and dischargingthe sample into a container, which is optionally the container CE, (2)drawing up into the solid support, at the same end, a solutioncomprising a compound conjugated to a label, called conjugate solutionCS, the conjugate solution C being contained in a container calledcontainer CC, allowing the conjugate solution CS to contact anddischarge into a container, which is optionally the container CC, (3) ifthe label uses a detection substrate, drawing up into the solid support,at the same end, a solution comprising a detection substrate with whichthe label will react, called substrate solution SS, the substratesolution SS being contained in a container called container CS, leavingin contact and discharging the substrate solution into a container,which is optionally the container CS, and (4) measuring the transmittedsignal.
 19. The detection or quantification process as claimed in claim18, wherein the container CE, the container CC and the container CS arecontained in the same test strip.
 20. The detection or quantificationprocess as claimed in claim 13, wherein the test sample to be analyzedis a sample of biological, food or environmental origin.